The broad long-term objective of the proposed research is to further our understanding of the molecular mechanisms used by DNA polymerase beta to catalyze the accurate synthesis of DNA. The Specific Aims are to determine if amino acid residues that are critical for fidelity of DNA polymerase Beta re located throughout the protein, to understand the molecular basis of infidelity of polymerase Beta mutator mutants, and to characterize the Pol Beta mutator mutants in vivo. DNA polymerase Beta has a central function in BER, which is a key repair pathway that is responsible for removing up to 10,000 DNA lesions per cell per day. Pol Beta also functions in meiosis. If an error is committed by Pol Beta during either of these processes, it has the potential to result in a mutation that can ultimately lead to human disease. Pol Beta has been implicated carcinogenesis by the demonstration that the Pol Beta locus is mutated in human colon and human prostate carcinomas; one of the mutations results in a protein that has a dominant negative effect on BER in mammalian cells. Therefore, results of mechanistic studies of Pol B will further our understanding of the molecular basis of mutation and have the potential to contribute directly to our understanding of the etiology of significant human diseases, including cancer. Our approach includes identifying and characterizing DNA polymerase Beta mutants using a genetic screen we developed, and analyzing these mutants biochemically and structurally. We also will characterize the phenotypes of polymerase Beta mutants in vivo.